The overall goal of the proposed research is to understand the means by which the obligately intracellular protozoan, Toxoplasma gondii can gain entrance to and live within the wide range of cells that it infects. Most of its host cells are non-phagocytic, and there is abundant evidence that T. gondii is both motile and can enter cells by active penetration. In this proposal I will concentrate on the analysis of the role of contractile proteins and cytoskeleton in the process of parasite movement and infection. It may also be possible to apply what is learned about the cytoskeleton of T. gondii and the relation of this intracellular parasite to the host cell's cytoskeleton to the analysis of other protozoan parasites such as Plasmodia, Leishmania or Trypanosomes. This research will be pursued utilizing cell cultures of fibroblasts and other relevant cells such as neural cells and mutant host cells which T. gondii can infect. The techniques I propose to employ are essentially those of cell biology. Immunofluorescence and immunoelectronmicroscopy will be used in conjunction with polyclonal antisera and monoclonal antibodies which react with components of the cytoskeleton and contractile proteins to localize these in the parasite and in the host cell. Cytoskeletal components of T. gondii will be extracted and characterized by immunoprecipitation, chromatography and electrophoresis. Monoclonal antibodies, some of which I have already produced, will be made against parasite and antigens. These will be screened for antibodies which react with T. gondii cytoskeleton, inhibit infection in cell culture, or produce immunofluorescence patterns suggestive of cytoskeleton or apical organelles. It may be possible to utilize appropriate monoclonal antibodies to select mutant parasites that are defective in a receptor or effector mechanism for cell penetration. In the long term we hope that the application of the techniques of cell biology will allow us to understand the mechanisms of movement and penetration of T. gondii and will lead to strategies for successful prevention of neonatal toxoplasmosis. In addition, the characterization of specific antigens associated with parasite penetration may allow development of serologic tests for the diagnosis of acute disease.